Iron is required by virtually all cells. The long-range goals of this proposal are to unravel the molecular mechanism of iron uptake in eukaryotic cells and determine how this uptake system is regulated. Using a combined genetic and biochemical approach, iron uptake in the yeast Saccharomyces cerevisiae will be analyzed. In yeast, iron uptake is a two- step process: extracellular Fe(III) is reduced to Fe(II) by a ferrireductase located in the plasma membrane. The Fe(II) product is then transported into the cell by an Fe(II) transporter. Uptake of transferrin- bound iron by human cells also appears to require a ferrireductase and an Fe(II) transporter. The proposed research should greatly increase our understanding of human iron uptake and the genetic defects responsible for iron overload diseases. This research is vital because the genes that encode the ferrireductase and iron transporter have not been isolated from any eukaryote. The molecular and genetic techniques available to study S. cerevisiae provide a unique opportunity to identify and characterize these genes and their products. Two genes, GEF1 and GEF2, have been identified as being necessary for yeast iron uptake. The following specific aims are proposed to study these genes. 1) GEF1's role in iron uptake will be determined by assaying ferrireductase and iron transport activities in mutant cells, subcellular localization of the protein by immunofluorescence microscopy, and biochemical analysis of Xenopus laevis oocytes that express the GEF1 protein from injected RNA. 2) The yeast gene encoding the iron transporter will be identified genetically, isolated, and characterized by DNA sequencing. 3) The mechanism that regulates synthesis of the iron transporter will be defined by Northern hybridization and, after antibodies have been prepared, immunoblot analysis. DNA sequence elements in the iron transporter gene that are required for transcriptional regulation will be identified using gene fusions in which the transporter gene's promoter is fused to the E. coli beta-galactosidase (lacZ) gene. How heme biosynthesis and intracellular iron levels control transcription of the transporter will be examined by studying the regulation of transporter-lacZ gene fusions in cells in which these aspects of iron metabolism have been experimentally altered. 4) GEF2's role in controlling transcription of the transporter gene will be assessed by studying this regulation in gef2- mutants and by determining the wild type protein's subcellular location. The GEF2 gene will be isolated and characterized by DNA sequencing and the GEF2 protein will be analyzed genetically and biochemically for transcriptional activation activity and DNA binding.